The objectives of this research are to understand the mechanisms by which pyrimidines are alkylated and halopyrimidines dehalogenated. Several enzymes important in cancer and cancer chemotherapy are involved in pyrimidine alkylation reactions. These include thymidylate synthetase, dCMP hydroxymethylase, and the RNA (DNA) methylases. Considerable evidence indicated that these reactions proceed by a mechanism that involves formation of a covalent enzyme-dihydropyrimidine intermediate. Experiments are described which should demonstrate the existence of such an intermediate in both a mammalian thymidylate synthetase and dCMP hydroxymethylase. These experiments include purification by affinity chromatography kinetic analysis, and possibile isolation of this intermediate.